The scale is based on 122 variables, 104 of which are binary variables and can either be 0 for “no, not done or not reported” or 1 for “done or reported”. Based on the current assay reporting practices in the literature, it is often not possible to distinguish between a variable that has not been addressed and a variable that has not been reported.

The 104 variables can be divided in 14 thematic blocks, such as “in silico analysis” or “extensive field testing of environmental samples”, which are associated with different levels of the validation scale. For example, “in silico analysis” is regarded as a very basic requirement and hence associated with Level 1 assays. “Extensive field testing of environmental samples” is considered an advanced part of the validation process and is thus associated with Level 4 assays.

Each of the variable blocks contains variables which are already considered standard in assay validation and reporting (e.g. report primer and probe sequences) and variables which today are hardly ever reported/validated but are likely to have substantial effect on the results (e.g. pressure used for filtration). As proof of a rudimentary validation effort, we defined minimum criteria (MC) for each variable block, e.g. “primer (and probe) sequence reported” is the minimum criterion for the “target tissue PCR” variable block. For an assay to be placed at a certain level of the validation scale, it must fulfil the MC of all variable blocks associated with this level. The scale is additive; hence, Level 2 assays must fulfil the Level 2 MC in addition to the Level 1 MC.

Note that the placement on the validation scale is location-specific, such that if an assay is to be used in a different region from the one where it was validated, additional field trials will need to be carried out in the new region to ensure the same level of performance is achieved. If additional closely related species are present in the new region, this warrants additional specificity testing.